Mammalian spermatozoa are vulnerable to membrane damage resulting in motility loss due to spontaneous peroxidation of phospholipids by O2, which places an ultimate limit on their motile lifetime in the oviduct. Work in this grant period showed that peroxidation in human sperm, as reflected in time to motility loss, is highly variable and correlates with strongly superoxide dismutase (SOD) activity and that inactivation of human sperm glutathione peroxidase (GPX) drastically shortens time to motility loss. These results imply that lipid hydroperoxide ROOH, as well as superoxide radical (SO), plays a key role in peroxidation. Lipid hydroperoxides PL-OOH were not found; all the ROOH was found as free fatty acid hydroperoxide (R(OOH)COOH). These results imply that a phospholipase alpha2 is active with PL-OOH, and also indicates that there may be a PL-OOH-specific GPX. Both activities would eliminate PL- OOH, and the hypothesis underlying this proposal is that human sperm have these two lines of defense against peroxidative damage. The analytical techniques developed during this period have led to high resolution chromatographic techniques for glycolipids; and two glycolipids, one anionic and so capable of activating phospholipase alpha2, and one neutral and so capable of inhibiting phospholipase alpha2, have been completely characterized. From the combination of these results have come the three specific aims for this proposal to test the hypothesis. 1). To characterize the phospholipase alpha2 activity in human sperm with regard to substrate specificity and possible activation by R(OOH)COOH. 2) To characterize the remaining glycolipids of human sperm and examine the activating or inhibiting activity of the sperm glycolipids with regard to phospholipase alpha2., 3). To characterize the GPX activity in human sperm with regard to substrate specificity, possible isozymes, and regulatory activity of free fatty acids and sperm glycolipids. Successful pursuit of these aims depends heavily on the use of sensitive, high resolution thin layer chromatography techniques which have been developed for this project during the present grant period. It is anticipated that successful pursuit of these aims will clarify those reactions inflicting peroxidative damage on human sperm and defending them against this damage, thus defining their susceptibility to spontaneous peroxidation reactions which may be a major component of unexplained male infertility. It is expected that the results from this proposed research may thus provide a new modality to help alleviate this condition.